Interleukin-2 mutein expressed from mammalian cells

ABSTRACT

Glycosylated interleukin-2 muteins are described. A method of producing the muteins using mammalian cells is included. The muteins may be incorporated into pharmaceutical preparations useful for, e.g., cancer therapy.

RELATED APPLICATION

Related subject matter is disclosed in patent application Ser. No.09/080,080, entitled “IL-2 Selective Agonists and Antagonists,” filedMay 15, 1998 in the names of Shanafelt et al., the entirety of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field

This invention generally relates to the recombinant production oftherapeutic proteins. More specifically, the invention is directed toglycosylated interleukin-2 muteins which can selectively activate Tcells (PHA-blasts) and reduce activation of Natural Killer (“NK”) cells.

2. Background

The biological activity of a glycoprotein is dependent upon not only theintegral structure of the protein, but also the properties of theoligosaccharide covalently attached to the protein. By influencing thephysico-chemical and biological properties of proteins, oligosaccharidescan modulate the therapeutic effect of a glycoprotein pharmaceutical. Itis well recognized that glycosylation can affect solubility, resistanceto proteolytic attack and thermal inactivation, quaternary structure,activity, targeting, antigenicity, functional activity, and half-life ofthe protein. The role of oligosaccharide in determining the in vivoactivity of EPO and the half-life of tissue plasminogen activator hasbeen reported.

Proleukin® (interleukin-2) has been approved by the FDA to treatmelanoma and renal carcinoma. However, due to the toxic side effectsassociated with interleukin-2, there is a need for a less toxic IL-2mutein that allows greater therapeutic use of this interleukin. Althoughnon-glycosylated interleukin-2 has been produced in E. coli with fullbiological activity, proper refolding of the recovered protein and thepotential for altered pharmacokinetics have been areas of concern. It isknown that the purification of interleukin-2 derived from E. coilrequires the use of chaotropic and toxic agents such as guanidinechloride and urea. Thus it would be advantageous to produce glycosylatedIL-2 muteins in mammalian cells where the use of harsh reagents can beavoided.

U.S. Pat. No. 5,417,970 to Roskam et al. (May 23, 1995), incorporatedherein by reference, discloses a wild type IL-2 preparation. Theabove-cited related application of Shanafelt et al. discloses IL-2muteins and states that the muteins may be expressed in a variety ofcells, including microbial, plant, and animal cells, including mammaliancells. We have now found a way to make such IL-2 muteins in glycosylatedform from mammalian cells. The characterization and details for making apreferred IL-2 mutein are described below.

SUMMARY OF THE INVENTION

We have developed a method for the production of glycosylated IL-2muteins from mammalian cells. Preferably the cell host is CHO cells, butthe production can be done with other cell hosts including HKB (see U.S.patent application Ser. No. 09/209,920 to Cho filed Dec. 10, 1998,incorporated herein by reference), myeloma, and 293S cells. Theproduction medium is preferably a chemically-defined medium free ofplasma protein supplements.

This invention is illustrated with a specific glycosylated polypeptidecomprising a human IL-2 mutein numbered in accordance with wild-typeIL-2 wherein said human IL-2 is substituted at position 88 witharginine, whereby said mutein preferentially activates T cells over NKcells. The preferred mutein is designated IL-2N88R, using conventionalterminology to describe the amino acid substitution of asparagine (N)with arginine (R) at position 88 of wild type IL-2. The nomenclature ofthe oligosaccharide structures is as described (Fukuda et al, 1994).Mammalian glycosylation patterns are well known and are described inFukuda et al. (1994), incorporated herein by reference.

This mutein exhibits essentially wild-type IL-2 activity on T cells.This invention is also directed to a method of treating a patientafflicted with an IL-2 treatable condition by administering atherapeutically effective amount of a human IL-2 mutein numbered inaccordance with wild-type IL-2 having PHA-blast activating activity buthaving reduced NK cell activating activity. This method is applicablewherein the IL-2 treatable condition is HIV, cancer, autoimmune disease,infectious disease, vaccine adjuvant in cancer vaccine and conventionalvaccine therapy for immune stimulation in the elderly or otherwiseimmunocompromised, as well as in human SCID patients, or othertherapeutic application requiring stimulation of the immune system.

BRIEF DESCRIPTION OF THE FIGURE

The FIGURE is a schematic diagram of the IL-2N88R expression vectorshowing the sites of the cytomegalovirus early promoter sequence(CMVe/p), the polyadenylation signal sequence (pA), and thedihydrofolate reductase (DHFR) sequence.

EXAMPLE 1

Development of Stable, High-producing CHO Cell Lines That ExpressIL-2N88R

Stable production cell lines that secrete high quantities of theIL-2N88R mutein were developed by transfecting CHO(dhfr-) cells with theexpression vector shown in FIG. 1. The vector was constructed usingstandard recombinant DNA techniques. The expression vector containsdiscrete expression cassettes for the IL-2N88R gene and the amplifiableand selectable gene DHFR (dihydrofolate reductase). The IL-2N88R genecodes for a protein having the amino acid sequence given by SEQ ID NO:1.About 1×10⁶ CHO (Chinese hamster ovary) cells were transfected with 10ug of pBC1N88R (see FIGURE) using LIPOFECTIN™ reagents (Life Technology,Bethesda, Md.) according to manufacturer's instructions. The cells werethen selected in the presence of 50 nM methotrexate and grown in DME/F12media deficient in thymidine and hypoxanthine plus 5% dialyzed fetalbovine serum. Cell populations were screened for IL-2N88R productionwith a commercial ELISA kit (R & D Systems). The high producingpopulations were further selected in media containing increasingconcentrations of methotrexate (100 to 400 nM methotrexate) and screenedfor the production of IL-2N88R. Limiting dilution cloning was thenapplied to derive clones with high and stable productivity. The cloningwas done in the absence of methotrexate using standard tissue culturetechniques. Mammalian cell culture techniques are well known anddisclosed in Freshey (1992), Mather (1998), Hu et al. (1997), andKonstantinov et al (1996), each of which are incorporated herein byreference.

EXAMPLE 2

Serumfree Production of IL-2N88R in a Perfusion Bioreactor

Continuous production of IL-2N88R was done by continuous perfusionfermentation. A 19-liter Wheaton fermenter was inoculated with a stableCHO cell line at 2×10⁶ cells/ml and perfused at a medium exchange rateof 5 liters/day. The production medium was a DME/F12-based mediumsupplemented with insulin (10 ug/ml) and FeSO₄.EDTA (50 uM). The celldensity was maintained at 4×10⁶ cells/ml. The average daily yield of thefermenter was ˜200 mg/day. The production IL-2N88R was stably maintainedfor 30 days.

EXAMPLE 3

Carbohydrate Analysis of IL-2N88R Produced from CHO Cells

IL-2N88R produced from CHO cells was purified using standardchromatography techniques involving ion exchange, reverse phase, andsize exclusion chromatography. The oligosaccharide structures ofIL-2N88R were characterized using glycosidases and matrix-assisted laserdesorption-ionization mass spectrometry (MALDI-MS).

IL-2N88R was analyzed either directly or after sequential digestion withsialidase, beta-galactosidases and O-glycanase on a MALDI spectrometerin linear mode. The observed molecular mass was compared with calculatedmolecular mass and the oligosaccharide structures were identified.

The total oligosaccharide pool was released with chemical hydrazinolysisand oligosaccharide mapping was performed by high pH anion-exchangechromatography with a CarboPac PA1 column.

IL-2N88R was found to be glycosylated only with O-linked GalNAc,GalNAc-β-Gal, and GalNAc-β-Gal-α-NeuNAc, of which monosialylatedGalNAc-β-Gal was the major oligosaccharide. A minor O-glycosylation sitebeside the site at Thr-3 was also detected. A summary of theoligosaccharide structures found in IL-2N88R was shown in the Table.

TABLE Assignment of oligosaccharide structures found in IL-2N88RStructure GalNAc N-acetylgalactosamine GalNAc-β-GalN-acetylgalactosamine-β- galactose GalNAc-β-Gal-α-NeuNAcN-acetylgalactosamine-β-galactose-α-N- acetylneuraminic acid

Conclusion

As illustrated in the above examples, we have developed a method for theproduction of IL-2 muteins having a mammalian glycosylation pattern. Itis thought that the method may be used to easily produce any IL-2mutein, using a variety of mammalian cells.

The above examples are intended to illustrate the invention and it isthought variations will occur to those skilled in the art. Accordingly,it is intended that the scope of the invention should be limited only bythe claims below.

REFERENCES

Cho, M-Y (1998) U.S. patent application Ser. No. 09/209,920 filed Dec.10, 1998.

Freshey, R. I. (ed) (1992) Animal Cell Culture: A Practical Approach,2nd ed. IRL Press, Oxford, England.

Fukuda et al. (1994) Molecular Glycobiology, IRL Press, New York

Hu, W. S., et al. (1997) Large-scale Mammalian Cell Culture, Curr OpinBiotechnol 8: 148-153

Konstantinov, K. B., et al. (1996) Control of long-term perfusionChinese hamster ovary cell culture by glucose auxostat, Biotechnol Prog12: 100-109

Liu (1992) Trends in Biotechnology 10: 114-120

Mather, J. P. (1998) Laboratory Scaleup of Cell Cultures (0.5-50liters), Methods Cell Biology 57: 219-527

Roskam et al. U.S. Pat. No. 5,417,970 (May 23, 1995)

Shanafelt et al. (1998) U.S. patent application Ser. No. 09/080,080filed May 15, 1998.

1 1 133 PRT Homo sapiens 1 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr GlnLeu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu AsnGly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr PheLys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln CysLeu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala GlnSer Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg IleAsn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met CysGlu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn ArgTrp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130

What is claimed is:
 1. An interleukin-2 mutein having a mammalianglycosylation pattern, wherein the interleukin-2 mutein is numbered inaccordance with wild-type interleukin-2 and the asparagine at position88 of the wild type interleukin-2 is substituted with arginine.
 2. Themutein of claim 1 wherein the glycosylation is O-linked.
 3. The muteinof claim 2 wherein the gylcosylation comprises O-linked GalNAc,GalNAc-β-Gal, and GalNAc-β-Gal-α-NeuNAc.
 4. A pharmaceutical preparationcomprising the mutein of claim 3 without a toxic solubilizing agent.